Plant development is a complex physiological and biochemical process requiring the coordinated expression of many genes. The production of new plant varieties with improved nutritional or disease-resistant traits can be achieved by modifying this coordinated pattern of gene expression. Recombinant DNA techniques have made it possible to alter the expression patterns of individual, specific plant genes without directly affecting the expression of other plant genes. In this way, the expression pattern of an individual gene can be either enhanced or diminished in the whole plant, in specific tissues, or in developmental stages. Thus, it is now routine to construct transgenes with defined promoters and terminators and express them in a variety of organisms.
However, there are some reports in the literature that some introduced transgenes do not have the expected expression patterns. These unexpected expression patterns are seen in organisms as diverse as nematodes and plants. For example, some plants receiving transgenic copies of an endogenous gene under the control of a strong promoter, sometimes fail to accumulate mRNA for that gene. Furthermore, all mRNA from endogenous genes having sequence homology to the transgene also fail to accumulate mRNA, effectively eliminating the expression of the endogenous gene product. This was discovered originally when chalcone synthase transgenes in petunia caused suppression of the endogenous chalcone synthase genes (Napoli et al (1990) Plant Cell 2:279-289).
The phenomenon was referred to as “cosuppression” since expression of both the endogenous gene and the introduced transgene were suppressed (for reviews see Vaucheret et al (1998) Plant J 16:651-659; and Gura (2000) Nature 404:804-808). Cosuppression technology constitutes the subject matter of U.S. Pat. No. 5,231,020 which issued to Jorgensen et al on Jul. 27, 1999. Cosuppression is also referred to as “gene silencing” or post-transcriptional gene silencing (PTGS) by plant biologists, “RNA interference” by those studying worms and flies (Montgomery and Fire (1998) TIG 14:255-258; Fire et al (1998) Nature 391:806-811; Hammond et al (2000) Nature 404:293-296; and PCT Application No. WO 99/32619 published Jul. 1, 1999), and “quelling” by researchers working with fungi (Romano and Macino (1992) Mol Microbiol 6:3343-3353).
The mechanisms by which the expression of a specific gene is inhibited by either antisense or sense RNA genes are not clearly understood and the frequencies of obtaining the desired down regulation in a transgenic plant are generally low and vary with the gene, the strength of its promoter and specificity, the method of transformation, and the complexity of transgene insertion events. (Grant (1999) Cell 96:303-306; and Selker (1999) Cell 97:157-160.)
The speculation is that PTGS is an ancient self-defense mechanism evolved to combat infection by viruses and transposons. It appears that this pathogen-derived resistance is triggered by the presence in the host's cells of double-stranded RNA (dsRNA) or some other aberrant nucleic acid, which are indicative of a viral assault. Normally, the RNA moving freely around a cell should be single-stranded messenger RNA (mRNA) which is the intermediate between host genes and the proteins they encode. When the aberrant RNA invades then any mRNAs matching the invading nucleic acid's sequence are shut down. If the trigger is homologous to part of the host's genetic sequence, then both the host and viral genes are silenced (Baulcombe (1996) Plant Cell 8:1833-1844). WO 99/15682 which published on Apr. 1, 1999 and WO 98/36083 which published on Aug. 20, 1998 describe gene silencing materials and methods. These publications describe, inter alia, the silencing of plant genomic gene expression by introducing expression constructs containing plant viral nucleic acid sequences coupled to whole, or partial, gene sequences homologous to the target genes to be silenced.
WO 99/53050, which published on Oct. 21, 1999, describes chimeric constructs encoding RNA molecules directed towards a target nucleic acid which are comprised of sense and antisense sequences, such that the expressed RNA is capable of forming an intramolecular double-stranded RNA structure. The expression of these RNA in transgenic organisms results in gene silencing of the all homologous target nucleic acid sequences within the cell.
U.S. Pat. No. 5,942,657, issued to Bird et al on Aug. 25, 1999, and WO 93/23551, which published on Nov. 25, 1993, describe coordinated inhibition of plant gene expression in which two or more genes are inhibited by introducing a single control gene having distinct DNA regions homologous to each of the target genes and a promoter operable in plants adapted to transcribe from such distinct regions RNA that inhibits expression of each of the target genes.
The present invention describes the use of suitable DNA sequences or RNA sequences derived therefrom, as is discussed below, in ways which here-to-fore have not been previously described. These sequences, and their reverse complements, can be used to reduce the expression of any endogenous genomic sequence that shares substantial similarity to nucleic acid fragment which is in proximity to the DNA sequence or RNA sequence derived therefrom. The details of this phenomenon are described herein.